CN117337327A

CN117337327A - Nanometer train for single molecule detection

Info

Publication number: CN117337327A
Application number: CN202280035664.4A
Authority: CN
Inventors: 张丕明; 张昕岳; 雷明
Original assignee: Universal Sequencing Technology Corp
Current assignee: Universal Sequencing Technology Corp
Priority date: 2021-04-24
Filing date: 2022-04-25
Publication date: 2024-01-02
Also published as: WO2022226426A1; US20240052401A1

Abstract

In one aspect, provided herein is an improved nanotrain for use in conjunction with a nanopore device for single molecule detection. Methods of making and using the nanotrains are also provided. The present disclosure relates to one-dimensional water-soluble nanoarrays, referred to herein as nanotrains, for single molecule detection of analytes, and methods of making the nanoarrays.

Description

Nanometer train for single molecule detection

Incorporated by reference

The present application claims priority and benefit from U.S. provisional application No. 63/179,186, filed 24 at 2021, 4, which is incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates to one-dimensional water-soluble nanoarrays (nanoarrays), referred to herein as nanotrains (nanotrain), for single molecule detection of analytes, and methods of making the arrays.

Background

Most disease states involve or disrupt multiple biochemical pathways, and quantification of various biomarkers is generally necessary for a comprehensive diagnosis and understanding of the disease. For example, a patient's miRNA profile may be a diagnostic feature that requires quantification of multiple mirnas. ¹ Furthermore, the discovery of biomarkers by different protein molecules is measured simultaneously ² Early detection of cancer, disease monitoring and personalized cancer treatment are of paramount importance. Detection of the protein marker panel may minimize false positives and false negatives in cancer diagnosis that may be caused by measuring a single biomarker. During the pandemic of new coronapneumonitis, multiplex assays have been used to evaluate different anti-SARS-CoV-2 antibodies for enhancing clinical sensitivity. ^3,4

Well-known multiplex detection techniques are microarrays in which molecular probes are attached to a solid surface ^5-8 And microbeads ^9-12 The method comprises the steps of carrying out a first treatment on the surface of the However, signal readout relies primarily on optical or electrochemical methods at ensemble-average level (ensemble-average level). A water-soluble 2D nanoarray has been developed to detect RNA detection using DNA self-assembly ^13,14 And proteins ¹⁵ It uses Atomic Force Microscopy (AFM) to examine binding events. However, as a single molecule technique, AFM requires trained personnel to make measurements and interpret the data. Furthermore, the cost and time consumption of AFM testing can be another factor limiting its use in clinical diagnostics.

Nanopore technology has been developed for nucleic acid sequencing and molecular detection over the last two decades. Nanopores are holes of nanometer diameter and depth assembled from biological materials or fabricated from inorganic materials. ¹⁶ It can be used as ion flow and organismNanofluidic channels for molecular transport. When a charged molecule is driven electrophoretically through a nanopore, it modulates ion current by partially blocking ion flow. Thus, the current blocking signal is recorded and used to identify the molecule and even its structural subunits. The nanopore is a single-molecule sensor for detecting DNA ¹⁷ 、RNA ^18-20 Protein ^21,22 Polysaccharide ^23-26 And other molecules. Since the charge is uniformly distributed along its phosphate backbone under physiological conditions, DNA can be efficiently translated through the nanopore. However, many molecules are uncharged, or sum to zero, which would make electrophoretic translocation inefficient. Thus, there is a need for improved supports (carriers) incorporating nanopore technology.

Drawings

Fig. 1 shows an exemplary DNA nanotrain carrying different molecular loads that when translocated through a nanopore device can cause fluctuations in ion current.

Fig. 2A-2D show an exemplary process of loading a load into a DNA nanotrain.

Fig. 3: an exemplary route to synthesize DNA nanovehicles and their attachment to microbeads to assemble DNA nanovehicles.

Fig. 4A-4B: (FIG. 4A) an exemplary pathway for the synthesis of DNA compartment (carriage) components; (fig. 4B) an exemplary pathway for the synthesis of polyethylene glycol linkers (linker) for assembling DNA nanotrains.

Fig. 5 shows an exemplary synthesis cycle for synthesizing DNA nanotrains on a solid substrate.

Fig. 6 shows an exemplary modified DNA structure for loading affinity molecules into the cars of a nanotrain.

Fig. 7: exemplary pathways for linking aptamers to modified DNA containing amino-modified thymidine.

Fig. 8A-8B: (FIG. 8A) polyethylene glycol functionalized with an azide at its 3' -end ₃₆ (PEG ₃₆ ) A pathway to modify the DNA compartment; (FIG. 8B) PEG functionalized with DBCO at its 5' -end ₂₄ The pathway of the DNA compartment is modified.

Fig. 9A-9B: (FIG. 9A) preparation of the PEG ₆₀ Two connectors are connectedExemplary approaches to nanotrains of DNA cars; (FIG. 9B) by PEG ₂₄ A nano train of two DNA carriages connected by a linker.

Fig. 10: gel electrophoresis image of DNA nanotrains and their components.

Fig. 11: gel electrophoresis image of DNA nanotrains hybridized with their complementary parts.

Disclosure of Invention

In one aspect, there is provided a nanotrain comprising:

a plurality of linearly arranged single stranded DNA compartments, each compartment having a unique sequence;

a plurality of complementary DNA sequences, each complementary DNA sequence being pre-designed to be complementary to a single-stranded DNA compartment, and each complementary DNA sequence hybridizing to its complementary single-stranded DNA compartment;

a plurality of affinity molecules for capturing one or more targets, wherein each affinity molecule is configured to be linked to a complementary DNA sequence; and

a plurality of flexible linkers connecting every two adjacent single stranded DNA cars, wherein each flexible linker is connected at a first end to the 5 '-end of a first single stranded DNA car and at a second end to the 3' -end of a second single stranded DNA car.

In some embodiments, each single stranded DNA compartment and each complementary DNA sequence is independently selected from the group consisting of a heterologous nucleic acid (XNA), a Peptide Nucleic Acid (PNA), a Locked Nucleic Acid (LNA), and a cyclohexenyl nucleic acid (CeNA), wherein the length of each single stranded DNA compartment and each complementary DNA sequence preferably ranges from 6 to 1000 bases.

In some embodiments, each complementary DNA sequence is modified to have a functional group that is pre-designed to attach an affinity molecule thereto, wherein preferably the functional group is selected from the group consisting of an amine, thiol, azide, alkyne, cycloalkyne, or tetrazine.

In some embodiments, each affinity molecule is independently selected from one or more of a nucleic acid, XNA, aptamer, ligand, antibody fragment, antigen, nanobody, affibody, protein, and/or carbohydrate.

In some embodiments, each affinity molecule comprises a microparticle, such as a magnetic bead, an amine, a thiol, an azide, an alkyne, a cycloalkyne, and/or a tetrazine.

In some embodiments, the one or more targets are selected from the group consisting of multiplex protein markers, single Nucleotide Polymorphisms (SNPs), DNA and RNA mutations, structural variations in the genome, drug molecules, antibodies, antigens, and glycans.

In some embodiments, each single stranded DNA compartment further comprises a protein molecule carrying at least one functional group that is orthogonal to those found in natural amino acids, such as an oxamine (oxamine), hydrazine, aldehyde, azide, alkyne, cycloalkyne, alkene, or tetrazine.

In some embodiments, each single stranded DNA compartment further comprises a charged polysaccharide bearing a functional group, such as a thiol, azide, alkyne, cycloalkyne, tetrazine, or oxamine.

In some embodiments, each flexible linker comprises a polypeptide having the structure:

wherein n=2 to 200; w=1 to 10; z=0 to 10

X= O, NH or ONH;

r=h, amine, thiol, azide, alkyne, cycloalkyne, tetrazine, carboxylate, hydroxyl, alkyl, alkene, guanidine, or glycan, selenium;

r' =azide, alkyne, cycloalkyne, tetrazine or aldehyde;

r "=h, azide, alkyne, cycloalkyne, cyclooctene, tetrazine or aldehyde.

In some embodiments, each flexible linker comprises polyethylene glycol having the following structure:

wherein n=2 to 500;

x = an amine, maleimide, vinyl sulfone, cyclooctene, thiol, azide, alkyne, cycloalkyne, tetrazine, oxamine, carboxylate, or aldehyde;

y = amine, maleimide, vinyl sulfone, thiol, azide, alkyne, cycloalkyne, tetrazine, oxamine, carboxylate, or aldehyde.

In some embodiments, the nanotrain may further comprise a neutral tail.

In some embodiments, the nanotrain may further comprise magnetic beads.

Another aspect relates to a system for single molecule detection, comprising:

any of the nanotrains disclosed herein,

a nanopore through which the nanotrain translocates, wherein the nanopore is formed of a biological, organic, inorganic, natural or synthetic material, and the nanopore has a pore size and thickness in the range of 2 to 1000nm, preferably 2 to 50 nm; wherein optionally a first pair of electrodes is embedded within the nanopore for measuring current, voltage and/or capacity;

a nanopore device having a cis reservoir (reservoir) and a trans reservoir separated by a membrane in which the nanopore is embedded;

a bias voltage applied between the cis reservoir and the trans reservoir through a second pair of electrodes;

means for recording current, voltage or capacity fluctuations caused by translocation of the nanotrain through the nanopore; wherein the current, voltage or capacity fluctuations detect one or more targets captured by the affinity molecule; and

software for data analysis that identifies or characterizes the one or more targets.

In some embodiments, the system may include a plurality of nanotrains, wherein the nanopore device includes a plurality of nanotrainsA nanopore, preferably an array of nanopores, wherein preferably the nanopore device contains 10 to 10 ⁹ Individual nanopores, preferably 10 ³ To 10 ⁷ Individual nanopores, or more preferably 10 ⁴ To 10 ⁶ And a plurality of nanopores.

Another aspect relates to a method for single molecule detection, comprising:

a. providing a system as disclosed herein;

b. mixing a nanotrain with a sample comprising one or more targets, thereby forming a loaded nanotrain having the one or more targets captured via a plurality of affinity molecules;

c. optionally separating the loaded nanotrain from the sample, preferably via magnetic beads that can be removed after the separation step;

d. placing the loaded nanotrain into a nanopore device, preferably in a cis reservoir;

e. applying a bias voltage between the cis reservoir and the trans reservoir to translocate the loaded nanotrain through the nanopore; and

f. recording current, voltage or capacity fluctuations caused by the translocation of the loaded nanotrain through the nanopore; wherein the current, voltage or capacity fluctuations detect one or more targets captured by the affinity molecule.

In some embodiments, the method is for high throughput detection of multiple targets using a nanopore device comprising multiple nanopores (preferably an array of nanopores), wherein preferably the nanopore device contains from 10 to 10 ⁹ Individual nanopores, preferably 10 ³ To 10 ⁷ Individual nanopores, or more preferably 10 ⁴ To 10 ⁶ And a plurality of nanopores.

Also provided is a method of synthesizing a nanotrain, comprising:

a. providing a plurality of compartments and a flexible linker, wherein each compartment is a single stranded DNA compartment having a unique sequence, wherein each compartment has orthogonal functional groups attached to its 5 '-and 3' -ends, wherein each functional group is independently selected from amine, maleimide, thiol, vinyl sulfone, azide, alkyne, cycloalkyne, cyclooctene, or oxamine;

b. connecting the head car to the solid substrate via a cleavable linker forming a first end of the nanotrain;

c. connecting a first flexible connector to the head car;

d. connecting a first car to the first flexible connector;

e. connecting the nth flexible connector to the (N-1) th car, wherein N is an integer greater than or equal to 2;

f. connecting the nth car to the nth flexible connector;

g. connecting the (n+1) th flexible connector to the nth car;

h. repeating steps (e) - (g) until a desired number of cars are connected by flexible connectors;

i. connecting a neutral tail to a second end of the nanotrain; and

j. cleaving the nanotrain from the solid substrate at the cleavable linker.

Detailed Description

In some embodiments, provided herein is a negatively charged DNA molecule that is used as a carrier for nanopore-based protein detection. Protein molecules captured on double-stranded DNA can be detected by translocation through the nanopore. ^27,28

The present disclosure provides a one-dimensional nanoarray (referred to as a nanotrain) for sensing biomolecules using a nanopore device, wherein the nanopore is biological, synthetic, solid state, or a combination thereof, and the size of the nanopore is about 2nm to 1000nm, preferably 2nm to 50nm. Fig. 1 shows a DNA nanotrain carrying multiple molecular loads sensed by nanopores. When a nanotrain electrophoreses translocate through a nanopore, each of its supports prevents electrolyte flow, resulting in ion current fluctuations. The variation of the ion current is controlled by its amplitude (Δi) and duration (t _d ) To measure and characterize. These resistance pulse parameters and peak positions can be used to identify individual payloads. The nano train can be used for detecting multiple protein markers, single Nucleotide Polymorphisms (SNP), DNA and RNA mutations,Structural variations in the genome, drug molecules, antibodies, antigens, and glycans.

Fig. 2A shows a typical DNA nanotrain comprising a locomotive (single-stranded DNA, 101) and four consecutive DNA carriages (single-stranded DNA,102, 103, 104, 105) interconnected by a flexible linker (106) with a neutral-charged tail (107) at their ends. These DNA compartments can be distinguished from each other by their sequence. For example, for the detection of proteins, nanotrains are equipped with receptors conjugated to complementary DNA (108, 109, 110, 111) and attached by hybridization to magnetic beads (112) functionalized with single-stranded DNA (113) (fig. 2B). 113 may be fabricated in the same manner as 205 in fig. 3. 113 is complementary to 101. 113 may be DNA or an analog thereof, e.g., LNA, cyclohexenyl nucleic acid (CeNA). When the equipped nanotrain is mixed with the sample solution, it captures those cognate ligands (114, 115, 116, 117, fig. 2C). The nanotrain loaded with the cargo may be separated from the solution by immobilizing it on the wall of the vial by magnetic force, and then released by cleaving the single-stranded DNA fragments beside the magnetic beads using nucleases. Thus, the loaded nanotrain with double-stranded DNA heads (118, fig. 2D) is ready for nanopore analysis as described above.

In some embodiments, polyethylene glycol (PEG) may be used as a linker to join DNA carriages to form a nanotrain. With lambda DNA vector ²⁸ Or double-stranded DNA ²⁹ In contrast, the neutral and flexible PEG linker can promote the entrance of the nanotrain into the nanopore, increasing the capture rate of the nanopore. It also prolongs the residence time of the nanotrain in the nanopore, resulting in an increase in the electrical signal. PEG has been studied as a crowded molecule (crowding molecule) that increases event frequency and residence time. ³⁰

The embodiments described and claimed herein have many attributes and embodiments, including but not limited to those set forth, described or referenced in this disclosure. The embodiments described and claimed herein are not limited or restricted by the features or embodiments identified in the present disclosure, which are for illustrative purposes only and not for limiting purposes. Hereinafter, a DNA nanotrain will be exemplified.

Definition of the definition

Certain terms are defined below. Additional definitions are provided throughout the application.

As used herein, the articles "a" and "an" refer to one or more than one (e.g., to at least one) of the grammatical object of the article. When used with the term "comprising," the use of the word "a" or "an" may mean "one," but it is also consistent with the meaning of "one or more," at least one, "and" one or more than one.

As used herein, "about" and "approximately" generally mean an acceptable degree of error in the measured quantity, taking into account the nature and precision of the measurement. Exemplary degrees of error are within 20% (%) of the given range, typically within 10%, more typically within 5%. The term "substantially" means more than 50%, preferably more than 80%, most preferably more than 90% or 95%.

As used herein, the term "comprising" refers to compositions, methods, and their respective components that are present in a given embodiment, but may include unspecified elements.

As used herein, the term "consisting essentially of … … (consisting essentially of)" refers to those elements required for a given embodiment. The term allows for the presence of additional elements that do not materially affect the basic and novel or functional characteristics of the embodiments of the present disclosure.

The term "consisting of … …" refers to compositions, methods and their respective components as described herein, excluding any elements not listed in the description of the examples.

As used herein, the term "nanotrain" refers to a linear molecular structure that includes a series of recognition entities connected by a linking molecule. These identification entities act as rail cars carrying the load (analyte). Part of the backbone of a railway car may be made of DNA, RNA, peptide Nucleic Acids (PNAs), polypeptides, polysaccharides, antibodies, receptors, supramolecules, and any charged or uncharged, natural, modified or synthetic linear molecules, or combinations thereof. Part or all of the backbone moiety may be a recognition entity, or be in top of (top of) or conjugated with a recognition entity (e.g., probe, receptor, antibody, molecular tweezer, etc.). Specifically, if the car includes DNA, it is called a DNA nanotrain. The binding molecules are an essential part of a nanotrain, designed to generate a signal that is different from the signal of the payload portion of the train car, typically of different size (diameter) or other characteristics due to the different nanopore signals.

Various aspects of the disclosure are described in further detail below. Additional definitions are provided throughout the specification.

In one embodiment, provided herein is a pathway for synthesizing the DNA nanotrain shown in fig. 2A. First, a precursor of the DNA locomotive (101) was synthesized according to the procedure described in FIG. 3. The amino group of N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -4-hydroxy-4-methylpentanamide (201) is first reacted with triphenylchloromethane (TrCl) to give the amino protected product (202). Method of using Bergstrom ³¹ The compound is introduced into the 5' -end of the nucleoside as a traceless cleavable linker. For example, compound 202 is attached to the 5' -end of thymidine by reaction with diisopropyldichlorosilane in the presence of diisopropylethylamine and imidazole, and a nucleoside in DMF. The resulting compound is phosphonylated (phosphinylation) to give thymidine phosphoramidite containing a cleavable linker (203). Phosphoramidite (203) is incorporated into the 5' -end of DNA nanolocomotive (101) to form precursor (204) by automated DNA synthesis. The nano-locomotive was attached by its amino group to microbeads functionalized with N-hydroxysuccinimide (NHS) ester (205, fig. 3). The nano locomotive precursor also carries a disulfide bond (disulfide linker) to connect a DNA car at its 3' -end for assembling a DNA nano train.

In some embodiments, methods for preparing a nanotrain DNA component for assembling a DNA nanotrain are provided. FIG. 4A depicts the pathway of synthesizing a DNA compartment (102, 103, 104 and 105 as depicted in FIG. 2A) with a PEG linker with azide function at its 5 '-end and a disulfide at its 3' -end (303).In detail, a DNA compartment containing an alkylamine at its 5 '-end and a disulfide functional group at its 3' -end (301) is synthesized in a conventional automatic DNA synthesizer. By conjugation with azido-dPEG _n TFP ester (302) reaction, DNA 301 can be easily converted to azide-functionalized DNA 303. Similarly, fig. 4B shows the pathway of synthesizing PEG linkers for linking DNA cars in a DNA nanotrain. diamino-PEG (401) is first reacted with dibenzo [ b, f]Azacycloocta-5 (6H) -pentanoic acid, 11, -12-didehydro-delta-oxo-, 2, 5-dioxo-1-pyrrolidinyl ester (DBCO-C5-NHS-ester, 402) reacts to produce an amino-PEG linker (403) containing DBCO functionality at one end thereof. It is further reacted with 5-maleimide valerate-NHS ester (404) to produce a PEG linker (405) containing maleimide functionality at its other end. These two functional groups of the PEG linker allow for assembly of DNA nanotrains by orthogonal click chemistry.

In one embodiment, a synthetic cycle for assembling a nanotrain on microbeads is provided (fig. 5). Synthesis begins with a nano locomotive precursor attached to microbeads (205). First treated with TCEP to reduce the disulfide to thiol, then reacted with PEG linker 405 (step i), and treated with methyl iodide (CH) ₃ I) Blocking unreacted mercaptans. Next, the microbeads are reacted with the component 303 containing DNA compartment 102 and blocking reagent 2-azidoethanol (step ii) to complete the first synthesis cycle. By repeating steps i and ii, DNA carriages 103, 104 and 105 are respectively connected to the DNA nanotrain. The last step (iii) is to attach the azide-PEG tail (501) to the DNA nanotrain to complete the whole process. Then, use literature ³¹ The fluoride reagent reported in (c) cleaves the product from the microbeads.

In some embodiments, methods of loading different affinity molecules into a DNA compartment to detect a targeted analyte are provided. First, a set of DNA molecules are synthesized whose sequence is complementary to the sequence of the cars in the nanotrain to contain functional groups, such as amine, thiol or other functional groups, respectively (fig. 6). The functional group is located at the end of the complementary DNA (601) or at the internal DNA base (602). These functional groups serve to attach the affinity molecule to the DNA. For example, FIG. 7 depicts a pathway for attaching an aptamer to DNA. First, complementary DNA of the compartment is synthesized to contain internal amino-modified thymidine (701). The DNA was then reacted with 2,3,5, 6-tetrafluorophenyl 3- (2-azidoethoxy) propionate (702) to give azide-functionalized DNA (703). The aptamer functionalized with an amine is reacted with DBCO-C5-NHS-ester (402) to produce an aptamer containing DBCO functionality (704). Mixing the DNA 703 with the aptamer 704 allows the click reaction to occur spontaneously, resulting in a DNA-aptamer conjugate (705). The conjugates were loaded into DNA nanotrains by hybridization with the DNA carriage using complementary sequences.

In some embodiments, different analytes are sensed/detected alone or in a combination of two or more analytes, wherein the analytes include, but are not limited to, DNA, RNA, proteins, antibodies/antigens, carbohydrates, any biological analytes, organic analytes, and the like.

In some embodiments, a plurality of nanotrain molecular structures are constructed and used with a nanopore chip device containing a plurality of nanopores or a nanopore array. In some embodiments, the nanopore array contains 10 to 10 ⁹ A plurality of nanopores, preferably 10 ³ To 10 ⁷ Individual nanopores, or more preferably 10 ⁴ To 10 ⁶ And a plurality of nanopores.

In some embodiments, the nanopore chip device is designed and constructed as described in WO201707562, WO2018209286, and PCT/US20/042188 (all of which are incorporated herein by reference). The nanopores may be of any shape, such as those described herein. The nanotrains and their respective targets can be measured using an ion current blocking method or using electrodes embedded in the nanopores (e.g., the electrodes described therein).

Example

In one embodiment, a nanotrain comprising two DNA carriages may be prepared. For example, DNA compartment-1 has a sequence of 5'-GAT CTG ACA GTA GGT ACG CAT CAG GAC ATC/3AmMO/-3' (SEQ ID NO. 1) with an amino linker (3 AmMO) at the 3 '-end (CAR-1 amine-3'), and compartment-2 has 5'-/5AmMC6/GAT ACA GGC TGC ACC ATT AGC GAC GGG ATC-3' (SEQ ID NO. 2) with an amino acid linker (5 AmMC 6) at the 5 '-end (CAR-2 amine-5'), as shown in FIG. 8A-8B. CAR-1 amine-3' and azido- ₃₆ TFP ester (801) reaction to produce DNA scaffold containing PEG linker with 36 ethylene glycol units long and ending with azide at its 3' -end (fig. 8a, car-1PEG ₃₆ Azide-3'). Similarly, CAR-2 amine-5' and DBCO-> ₂₄ TFP ester (802) reaction to produce a DNA compartment containing a PEG linker 24 ethylene glycol units long and ending with DBCO at its 5' -end (CAR-2 PEG) ₂₄ DBCO-5', FIG. 8B). Finally, CAR-1PEG ₃₆ Azide-3' with CAR-2PEG ₂₄ DBCO-5' was mixed in aqueous solution, resulting in a nanotrain with two DNA compartments linked via a PEG linker of 60 ethylene glycol units (CAR-1 PEG ₆₀ CAR-2, fig. 9-a). Similarly, CAR-2PEG was conjugated to CAR-1C 6-azide-3' via CAR-1C 6-azide-3 ₂₄ DBCO-5' reacted in aqueous solution, assembled a nanotrain with two DNA compartments linked via a PEG linker of 24 ethylene glycol units (CAR-1 PEG ₂₄ CAR-2) (fig. 9-b). These desired products were verified by gel electrophoresis (fig. 10). Lane 1: CAR-1 amine-3'; lane 2: CAR-2 amine-5'; lane 3: gel purified CAR-1PEG ₃₆ Azide-3' (sample # 2); lane 4: gel purified CAR-1PEG ₃₆ Azide-3' (sample # 2); lane 5: precipitated CAR-2PEG ₂₄ DBCO-5'; lane 6: purified CAR-1PEG ₂₄ CAR-2; lane 7: for the production of CAR-1PEG ₆₀ Reaction mixture of CAR-2.

In some embodiments, the nanotrains described above carry complementary DNA portions. Examples include two DNA entities, 5'-GAT GTC CTG ATG CGT ACC TAC TGT CAG ATC-3' (CAR-1C) (SEQ ID No. 3) and 5'-GAT CCC GTCGCT AAT GGT GCA GCC TGT ATC-3' (CAR-2C) (SEQ ID No. 4) complementary to CAR-1 and CAR-2, respectively. Gel electrophoresis analysis shows that CAR-1PEG ₆₀ CAR-2 and CAR-1PEG ₂₄ The CAR-2 hybridizes efficiently to the complementary DNA entity,although they were assembled by PEG linkers with different lengths (fig. 11). Lane 1: CAR-1C; lane 2: CAR-2C; lane 3: CAR-1PEG ₆₀ CAR-2+car-1c+car-2C (ratio=1:1:1); lane 4: CAR-1PEG ₆₀ CAR-2+car-1c+car-2C (ratio=1:1.1:1.1); lane 5: CAR-1PEG ₆₀ CRA-2+car-1c+car-2C (ratio=1.1:1:1); lane 6: CAR-1PEG ₆₀ CAR-2+car-1C (ratio=1:1); lane 7: CAR-1PEG ₆₀ CRA-2+car-2C (ratio=1:1); lane 8: CAR-1PEG ₂₄ CAR-2+car-1c+car-2C (1:1:1); lane 9: CAR-1PEG ₆₀ CAR-2 reaction mixture; lane 10: CAR-2PEG ₂₄ DBCO-5'; lane 11: o' Range rule 20bp DNA ladder. In addition, these unhybridized residues can be easily separated from the product.

Additional embodiments:

systems for identifying and sensing single and multiple molecules:

a. a nanopore comprising a biological, organic or inorganic material and having a pore size and thickness in the range of 2 to 1000nm, preferably 2 to 50 nm;

b. a nanotrain comprising a plurality of cars (n > 1), each car having attached thereto an affinity molecule for capturing its respective target, wherein the cars comprise a charged head and a neutral tail interspersed with flexible linkers, and wherein the affinity molecules comprise, but are not limited to, nucleic acids, XNA, aptamers, ligands, antibodies, antibody fragments, antigens, nanobodies, affibodies, proteins, or carbohydrates, wherein the affinity molecules comprise, but are not limited to, amines, thiols, azides, alkynes, cycloalkynes, or tetrazines;

c. a nanopore device having two reservoirs separated by a membrane having embedded nanopores;

d. a bias voltage applied between the two reservoirs through a pair of electrodes;

e. means for recording current fluctuations caused by the nanotrain translocating the nanopore;

f. software for data analysis that identifies or characterizes target biomolecules;

g. a DNA compartment comprising a nucleic acid comprising DNA ranging from 6 to 1000 bases in length, RNA ranging from 6 to 1000 bases in length, which can form a duplex, triplex, quadruplex with itself or its complementary nucleic acid, wherein the nucleic acid and its complementary nucleic acid contain functional groups, including but not limited to amines, thiols, azides, alkynes, cycloalkynes, or tetrazines, inside or at the end that can link an affinity molecule to the compartment, and wherein the nucleic acid and its complement comprise natural nucleosides, non-natural nucleosides, or combinations thereof;

h. a DNA compartment having a heterologous nucleic acid (XNA) including, but not limited to, a Peptide Nucleic Acid (PNA), a Locked Nucleic Acid (LNA), a cyclohexenyl nucleic acid (CeNA), which XNA can form a duplex, triplex, quadruplex with itself or its complement, wherein the XNA and the complement XNA contain functional groups, including, but not limited to, amines, thiols, azides, alkynes, cycloalkynes, or tetrazines, inside or at the end that can attach an affinity molecule to the compartment, and wherein the XNA and the complement XNA include natural nucleosides, unnatural nucleosides, or combinations thereof;

i. a compartment comprising a protein molecule carrying at least one functional group orthogonal to those found in natural amino acids, including, but not limited to, oxamines, hydrazines, aldehydes, azides, alkynes, cycloalkynes, alkenes, or tetrazines;

j. the compartment of the above part b includes protein molecules carrying at least one functional group orthogonal to those present in natural amino acids, including, but not limited to, oxamines, hydrazines, aldehydes, azides, alkynes, cycloalkynes, alkenes, or tetrazines;

k. the compartment of the above part b comprises charged polysaccharides carrying functional groups including, but not limited to, thiols, azides, alkynes, cycloalkynes, tetrazines, or oxamines;

the flexible linker of the above part b comprises a polypeptide

Wherein n=2 to 200; w=1 to 10; z=0 to 10

X= O, NH or ONH;

r=h, amine, thiol, azide, alkyne, cycloalkyne, tetrazine, carboxylate, hydroxyl, alkyl, alkene, guanidine or glycan, selenium, but are not limited thereto;

r' =azide, alkyne, cycloalkyne, tetrazine or aldehyde, but is not limited to them;

r "=h, azide, alkyne, cycloalkyne, cyclooctene, tetrazine or aldehyde, but is not limited to them;

m. a flexible linker of the above part b, comprising polyethylene glycol, the flexible linker having the structure:

wherein n=2 to 500;

x = an amine, maleimide, vinyl sulfone, cyclooctene, thiol, azide, alkyne, cycloalkyne, tetrazine, oxamine, carboxylate, or aldehyde, but is not limited thereto;

x = an amine, maleimide, vinyl sulfone, thiol, azide, alkyne, cycloalkyne, tetrazine, oxamine, carboxylate, or aldehyde, but is not limited thereto;

n. the flexible linker of the above moiety b comprises a combination of the polypeptide of the above moiety m and the polyethylene glycol of the above moiety l.

A method of synthesizing a nanotrain on a solid substrate:

a. a method for synthesizing a nano train.

b. A cleavable linker at one end of the nanolocomotive through which the first component of the nanolocomotive is attached to the solid substrate.

c. Orthogonal functional groups attached to both ends of the car, wherein the functional groups are amines, maleimides, thiols, vinyl sulfones, azides, alkynes, cycloalkynes, cyclooctenes, oxamines, but are not limited thereto.

d. The synthetic cycle of sequentially incorporating the flexible linker and the car component into the nano locomotive is repeated.

e. Neutral tails are incorporated into the ends of the nanotrains.

f. The nanotrain is cleaved from the solid substrate.

Wherein the solid substrate comprises microbeads, nanobeads, planar substrates (flat substrates), but is not limited to them; and wherein the solid substrate comprises an inorganic material, a metal oxide, a polymeric material, an organic material, but is not limited thereto.

All publications, patents, and other documents mentioned herein are incorporated by reference in their entirety. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. While the present disclosure has been illustrated by a description of various embodiments and while these embodiments have been described in considerable detail, it is not the intention of the applicant to restrict or in any way limit the scope of the application. Additional advantages and modifications will readily occur to those skilled in the art. The disclosure, in its broader aspects, is therefore not limited to the specific details, the representative apparatus, devices, and methods and illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the spirit of applicant's general inventive concept.

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Claims

1. a nanotrain, comprising:

a plurality of linearly arranged single stranded DNA compartments, each single stranded DNA compartment having a unique sequence;

2. The nanotrain of claim 1, wherein each single stranded DNA compartment and each complementary DNA sequence is independently selected from the group consisting of heterologous nucleic acid (XNA), peptide Nucleic Acid (PNA), locked Nucleic Acid (LNA) and cyclohexenyl nucleic acid (CeNA), wherein preferably each single stranded DNA compartment and each complementary DNA sequence ranges from 6 to 1000 bases in length.

3. The nanotrain of claim 1, wherein each complementary DNA sequence is modified to have a functional group pre-designed to attach an affinity molecule thereto, wherein preferably the functional group is selected from the group consisting of amine, thiol, azide, alkyne, cycloalkyne, or tetrazine.

4. The nanotrain of claim 1, wherein each affinity molecule is independently selected from one or more of a nucleic acid, XNA, aptamer, ligand, antibody fragment, antigen, nanobody, affibody, protein, and/or carbohydrate.

5. The nanotrain of claim 1, wherein each affinity molecule comprises a microparticle, such as a magnetic bead, an amine, a thiol, an azide, an alkyne, a cycloalkyne, and/or a tetrazine.

6. The nanotrain of claim 1, wherein the one or more targets are selected from the group consisting of multiplex protein markers, single Nucleotide Polymorphisms (SNPs), DNA and RNA mutations, structural variations of the genome, drug molecules, antibodies, antigens, and glycans.

7. The nanotrain of claim 1, wherein each single stranded DNA compartment further comprises a protein molecule carrying at least one functional group orthogonal to those found in natural amino acids, such as an oxamine, hydrazine, aldehyde, azide, alkyne, cycloalkyne, alkene, or tetrazine.

8. The nanotrain of claim 1, wherein each single stranded DNA compartment further comprises a charged polysaccharide carrying a functional group, such as a thiol, azide, alkyne, cycloalkyne, tetrazine, or oxamine.

9. The nanotrain of claim 1, wherein each flexible linker comprises a polypeptide having the structure:

wherein n=2 to 200; w=1 to 10; z=0 to 10

X= O, NH or ONH;

r' =azide, alkyne, cycloalkyne, tetrazine or aldehyde;

r "=h, azide, alkyne, cycloalkyne, cyclooctene, tetrazine or aldehyde.

10. The nanotrain of claim 1, wherein each flexible linker comprises polyethylene glycol having the structure:

wherein n=2 to 500;

11. The nanotrain of claim 1, further comprising a neutral tail.

12. The nanotrain of claim 1, further comprising magnetic beads.

13. A system for single molecule detection, comprising:

the nanotrain according to any of claims 1-12,

a nanopore device having a cis reservoir and a trans reservoir separated by a membrane in which the nanopore is embedded;

14. The system of claim 13, wherein the one or more targets are selected from the group consisting of multiplex protein markers, single Nucleotide Polymorphisms (SNPs), DNA and RNA mutations, structural variations of the genome, drug molecules, antibodies, antigens, and glycans.

15. The system of claim 13, comprising a plurality of nanotrains, wherein the nanopore device comprises a plurality of nanopores, preferably an array of nanopores, wherein preferably a nanopore device contains 10 to 10 ⁹ Individual nanopores, preferably 10 ³ To 10 ⁷ Individual nanopores, or more preferably 10 ⁴ To 10 ⁶ And a plurality of nanopores.

16. A method for single molecule detection, comprising:

(a) Providing a system according to any one of claims 13-15;

(b) Mixing a nanotrain with a sample comprising one or more targets, thereby forming a loaded nanotrain having the one or more targets captured via a plurality of affinity molecules;

(c) Optionally separating the loaded nanotrain from the sample;

(d) Placing the loaded nanotrain into a nanopore device, preferably in a cis reservoir;

(e) Applying a bias voltage between the cis reservoir and the trans reservoir to translocate the loaded nanotrain through the nanopore; and

(f) Recording current, voltage or capacity fluctuations caused by the translocation of the loaded nanotrain through the nanopore; wherein the current, voltage or capacity fluctuations detect one or more targets captured by the affinity molecule.

17. The method of claim 16, wherein the method is for high throughput detection of multiple targets, wherein the nanopore device comprises a plurality of nanopores, preferably an array of nanopores, wherein preferably the nanopore device comprises 10 to 10 ⁹ Individual nanopores, preferably 10 ³ To 10 ⁷ Individual nanopores, or more preferably 10 ⁴ To 10 ⁶ And a plurality of nanopores.

18. A method of synthesizing a nanotrain, comprising:

(a) Providing a plurality of compartments and a flexible linker, wherein each compartment is a single stranded DNA compartment having a unique sequence, wherein each compartment has orthogonal functional groups attached to its 5 '-and 3' -ends, wherein each functional group is independently selected from amine, maleimide, thiol, vinyl sulfone, azide, alkyne, cycloalkyne, cyclooctene, or oxamine;

(b) Connecting the head car to the solid substrate via a cleavable linker forming a first end of the nanotrain;

(c) Connecting a first flexible connector to the head car;

(d) Connecting a first car to the first flexible connector;

(e) Connecting the nth flexible connector to the (N-1) th car, wherein N is an integer greater than or equal to 2;

(f) Connecting the nth car to the nth flexible connector;

(g) Connecting the (n+1) th flexible connector to the nth car;

(h) Repeating steps (e) - (g) until a desired number of cars are connected by flexible connectors;

(i) Connecting a neutral tail to a second end of the nanotrain; and

(j) Cleaving the nanotrain from the solid substrate at the cleavable linker.